K-RasG12D Has a Potential Allosteric Small Molecule Binding Site

Biochemistry. 2019 May 28;58(21):2542-2554. doi: 10.1021/acs.biochem.8b01300. Epub 2019 May 14.


KRAS is the most commonly mutated oncogene in human cancer, with particularly high mutation frequencies in pancreatic cancers, colorectal cancers, and lung cancers [Ostrem, J. M., and Shokat, K. M. (2016) Nat. Rev. Drug Discovery 15, 771-785]. The high prevalence of KRAS mutations and its essential role in many cancers make it a potentially attractive drug target; however, it has been difficult to create small molecule inhibitors of mutant K-Ras proteins. Here, we identified a putative small molecule binding site on K-RasG12D using computational analyses of the protein structure and then used a combination of computational and biochemical approaches to discover small molecules that may bind to this pocket, which we have termed the P110 site, due to its adjacency to proline 110. We confirmed that one compound, named K-Ras allosteric ligand KAL-21404358, bound to K-RasG12D, as measured by microscale thermophoresis, a thermal shift assay, and nuclear magnetic resonance spectroscopy. KAL-21404358 did not bind to four mutants in the P110 site, supporting our hypothesis that KAL-21404358 binds to the P110 site of K-RasG12D. This compound impaired the interaction of K-RasG12D with B-Raf and disrupted the RAF-MEK-ERK and PI3K-AKT signaling pathways. We synthesized additional compounds, based on the KAL-21404358 scaffold with more potent binding and greater aqueous solubility. In summary, these findings suggest that the P110 site is a potential site for binding of small molecule allosteric inhibitors of K-RasG12D.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Allosteric Site / drug effects*
  • Catalytic Domain
  • Cell Line, Tumor
  • Drug Discovery / methods
  • Escherichia coli / metabolism
  • HEK293 Cells
  • Humans
  • MAP Kinase Signaling System / drug effects*
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Mutant Proteins / antagonists & inhibitors
  • Mutant Proteins / chemistry
  • Phosphatidylinositol 3-Kinases / metabolism
  • Protein Binding
  • Protein Structure, Secondary
  • Proto-Oncogene Proteins B-raf / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism
  • Proto-Oncogene Proteins p21(ras) / antagonists & inhibitors*
  • Proto-Oncogene Proteins p21(ras) / chemistry*
  • Proto-Oncogene Proteins p21(ras) / metabolism
  • Transfection


  • Mutant Proteins
  • Proto-Oncogene Proteins B-raf
  • Proto-Oncogene Proteins c-akt
  • Proto-Oncogene Proteins p21(ras)